Bispecific CD19 CD37 CAR T cells. (A) Two bispecific second-generation CARs were constructed with a different order of the single-chain variable fragments: CAR-19-37 (top) and CAR-37-19 (bottom). (B) Activation of Jurkat reporter (NFAT-Luc) T cells transduced with different CAR constructs and cocultured with tumor cells. Luciferase activity was measured after 16 hours. CD3-CD28 beads: positive control. (C) Ex vivo expansion of CD3/CD28 bead-activated and target-stimulated T cells in 2 healthy donors for 30 days. (D) Cytotoxic capacity of bispecific CAR T cells was measured after overnight coculture with K562 targets transduced with CD37, CD19, or both at indicated E:T ratios. The cytotoxicity assay is representative of 2 independent experiments conducted with different healthy donors. (E) NSG mice were injected intravenously with 1 × 10⁶ JEKO-1 (CBG⁻GFP) cells and monitored by BLI for tumor burden over time. At day 0, mice were randomly assigned on the basis of tumor burden (BLI) to receive 2 × 10⁶ control T cells (UTD), CAR- 37, CAR-19, CAR-19-37, or CAR-37-19. All CAR T-cell groups were normalized to have the same % CAR⁺ cells and untransduced cells. Average flux (photons/s) of whole mice at different points is shown. Graph shows 1 experiment with 6 mice per group. (F) CAR T cells were enumerated in peripheral blood by flow cytometry at the indicated points. Absolute counts of CAR T cells are shown as mean ± SEM.